Inhibiting MOG-antibody binding

ABSTRACT

The invention provides methods and compositions for inhibiting pathogenic binding of an pathogenic autoantibody to a myelin oligodendrocyte glycoprotein (MOG) autoantigen and screening for inhibitors of pathogenic binding of an autoantibody to a MOG autoantigen.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/384,036, filed Aug.26, 1999, now U.S. Pat. No. 6,333,033 which claims the benefit of U.S.Provisional Application 60/097,953 filed Aug. 26, 1998, both of whichare incorporated herein by reference.

This invention was made with Government support under Grant No. AI43073, awarded by the National Insitutes of Health. The Government hascertain rights in this invention.

INTRODUCTION

1. Field of the Invention

The field of this invention is polypeptide autoantibody inhibitors andmethods of use thereof.

2. Background

Multiple sclerosis (MS) is a chronic relapsing remitting disorderdisease of the central nervous system that affects 350,000 Americansand, second to trauma, is the leading cause of disability among youngadults. MS is an immune-mediated disorder characterized pathologicallyby perivenular white matter infiltrates comprised of macrophages andmononuclear cells (inflammation), and destruction of the myelin sheathsthat insulate nerve fibers (demyelination).

Experimental allergic encephalomyelitis (EAE) in rodents has been themost widely employed model for testing of therapies for human MS. Thesetraditional disease models for MS generally have promoted the conceptthat MS is a T-cell-mediated disorder. However, the autoantigens thatserve as targets for the immune attack have not been identified and themolecular mechanisms implicated in myelin damage remain uncertain. Whileit is clear that CNS inflammation in EAE is initiated by autoagressiveT-cells that recognize myelin antigens in the context of class II-MHCmolecules, many of the models lack the early demyelinating component ofthe MS lesion. B-cell activation and antibody responses appear necessaryfor the full development of EAE and earlier studies on immune mediateddemyelination using myelinated cultures of CNS tissue have implicatedhumoral factors as effector mechanisms. Thus, it is not surprising thatrodent EAE has not been a robust predictor of efficacy in humans asfundamental differences in the clinical course, pathology, andimmunologic response to myelin proteins distinguish rodent EAE fromhuman MS.

Recently a novel MS-like illness in an outbred nonhuman primate, thecommon marmoset Callithrix jacchus, has been defined. The marmoset EAEhas a prominent, MS-like early demyelinating component which requiresthe presence of myelin-specific autoantibodies, and has afforded anopportunity to understand the interactions between these antibodies andtheir target antigens on myelin. Characteristics of the model include:a. Mild clinical signs and a relapsing remitting course similar to MS;b. A primary demyelinating pathology with early gliosisindistinguishable from MS lesions (demyelinating plaques); c. Naturalbone marrow chimerism permitting successful adoptive transfer ofencephalitogenic (e.g. disease-inducing) T-cell clones and lines; d.Diversity of the encephalitogenic repertoire of T-cells reactive againstthe major myelin protein myelin basic protein (MBP); e. Differentdisease phenotypes resulting from immunization with different myelinconstituents: in contrast to whole myelin, immunization with MBPproduces a non-demyelinating form of EAE; f. Demonstration thatdemyelination is antibody-mediated but also requires an encephalitogenicT-cell response to facilitate autoantibody access to the nervous system;and, g. A key role of myelin oligodendrocyte glycoprotein (MOG) inplaque formation: adoptive transfer of anti-MOG antibody innon-demyelinating MBP-EAE reproduces fully developed MS-like pathology.

The highly immunogenic properties of MOG (<0.05% of total myelinprotein) may be related to its extracellular location on the outermostlamellae of the myelin sheath, where it is accessible to pathogenicantibody in the context of blood brain barrier disruption byencephalitogenic T-cells. The C. jacchus model permits preciseidentification of cellular and humoral immune responses that result inan MS-like lesion in a species with immune and nervous system genes thatare 90-95% homologous to humans. The relevance of this model to human MSis emphasized by the recent finding of strong T-cell and antibodyresponses to MOG in MS patients.

SUMMARY OF THE INVENTION

The present invention is directed to autoantibody inhibitors and methodsof use thereof. Accordingly, the invention provides methods andcompositions for inhibiting pathogenic binding of an autoantibody to anautoantigen and screening for inhibitors of pathogenic binding of anautoantibody to an autoantigen.

In one aspect, the present invention provides a composition comprising apeptide consisting of residues 28-36, 13-21, 62-74, 27-34 or 40-45 ofrat, human or marmoset MOG (SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:2,respectively). In a preferred embodiment the MOG polypeptide is directlyjoined at its N- and C-termini with other than natural human or marmosetMOG flanking residues.

In another aspect, the present invention provides a method of inhibitingpathogenic binding of a MOG specific autoantibody to MOG or animmunodominant epitope thereof.

In yet another aspect, the present invention provides a method ofdetecting autoantibodies in a tissue sample. In a preferred embodiment amethod of identifying autoantibodies against myelin/oligodendrocyteglycoprotein (MOG) within lesions of human MS and C. jacchus EAE, wherethey appear to be directly responsible for the disintegration of themyelin sheaths, is provided.

In a further aspect, the present invention provides a method ofscreening small molecules or candidate agents capable of binding to anautoantigen and thereby inhibit binding of an autoantibody. The methodcomprises contacting a solution comprising an autoantigen and anautoantibody, incubating under conditions sufficient to allow thereaction to reach equilibrium, and comparing the binding of theautoantibody in the absence of the small molecule inhibitor or candidateagent to the binding of the autoantibody in the presence of the smallmolecule inhibitor or candidate agent. In a preferred embodiment thesmall molecules specifically bind at least one immunodominant epitope ofthe autoantigen.

In yet another aspect of the invention there is provided a method ofinhibiting pathogenic binding of an autoantibody to an autoantigencomprising administering to a host subject to pathogenicautoantigen-autoantibody binding-mediated pathology an effective amountof a composition comprising a fragment of an antibody specific for theautoantigen sufficient to specifically bind the autoantigen andcompetitively inhibit the binding of an autoantigen-specificautoantibody to the autoantigen, wherein the fragment does not comprisea functional Fc portion of the autoantigen-specific antibody. In apreferred embodiment, the autoantigen-autoantibody binding is associatedwith a demyelinating disease of the central or peripheral nervoussystem. In a particular embodiment, the disease is associated withpathogenic autoantibody binding, such as MS, lupus, arthritis ordiabetes. In more particular embodiments, the autoantigen is a MOGautoantigen and the fragment is a F(ab′)₂ fragment.

In yet another aspect, the invention also provides methods of screeningfor an agent which modulates the binding of an autoantibody to anautoantigen. Such methods generally involve incubating a mixturecomprising the autoantibody or an auto antibody-specific bindingfragment thereof, the autoantigen, and a candidate agent underconditions whereby, but for the presence of said agent, the autoantibodyor fragment thereof specifically binds the autoantigen at a referenceaffinity; detecting the binding affinity of autoantibody or fragmentthereof to the autoantigen to determine an agent-biased affinity,wherein a difference between the agent-biased affinity and the referenceaffinity indicates that said agent modulates the binding of theautoantibody or fragment thereof to the autoantigen. In particularembodiments, the autoantibody or fragment thereof is a F(ab′)₂ fragment;the autoantigen comprises a MOG epitope; and/or the autoantigencomprises a MOG epitope consisting of residues 28-36, 13-21, 62-74,27-34 or 40-45 of rat, human or marmoset MOG (SEQ ID NO:1, SEQ ID NO:3and SEQ ID NO:2, respectively).

DETAILED DESCRIPTION OF THE INVENTION

The following description and examples are offered by way ofillustration and not by way of limitation.

The invention provides methods and compositions for inhibiting pathologyassociated with the binding of an autoantibody to a MOG polypeptide,such as occurs in MS. The general methods comprise the step ofadministering to a host, subject to a pathogenic MOGpolypeptide-autoantibody binding, an effective amount of a compositioncomprising a MOG polypeptide-specific antibody fragment not having afunctional Fc portion and sufficient to specifically bind the MOGpolypeptide and competitively inhibit the binding of the autoantibody tothe MOG polypeptide, whereby the pathology is inhibited. In a particularembodiment, the fragment is selected from the group consisting of Fv,F(ab′)₂, F(ab), F(ab)₂ or fragments thereof.

The compositions include pharmaceutical compositions comprising a MOGpolypeptide-specific antibody fragment sufficient to specifically bind anatural MOG polypeptide and competitively inhibit the binding of anautoantibody to the MOG polypeptide, wherein the fragment does notcomprise a functional Fc portion, and a pharmaceutically acceptablecarrier. The compositions may also comprise a MOG tolerogenic T-cellepitope which induces tolerance and acts synergistically with theantibody fragment to inhibit pathology.

In another embodiment, the invention provides methods and compositionsfor detecting the presence of an autoantibody bound to a firstautoantigen in a tissue. These methods generally comprise the steps ofcontacting the tissue with a second, labeled autoantigen underconditions wherein the autoantibody binds the second autoantigen to formfirst autoantigen-autoantibody-second autoantigen labeled complexes, andspecifically detecting the labeled complexes. The first and secondautoantigens are generally the same or at least include epitopes of thesame autoantigen. Preferred autoantigens include, but are not limited tomyelin oligodendrocyte glycoprotein (MOG), myelin associatedglycoprotein (MAG), myelin/oligodendrocyte basic protein (MOBP),Oligodendrocyte specific protein (Osp), myelin basic protein (MBP),proteolipid apoprotein (PLP), galactose cerebroside (GalC), glycolipids,sphingolipids, phospholipids, gangliosides and other neuronal antigens.

In yet another embodiment, the invention provides methods andcompositions for detecting MOG polypeptide-specific B-cells. Suchmethods generally comprise the steps of fractionating blood to obtain anunselected population of B-cells comprising rare MOGpolypeptide-specific B-cells, contacting the population with labeled MOGpolypeptides under conditions whereby the labeled MOG polypeptides bindsthe rare MOG polypeptide-specific B-cells to form labeled complexes ofthe labeled MOG polypeptides and the rare MOG polypeptide-specificB-cells, and specifically detecting the complexes.

In yet another embodiment, the invention provides methods andcompositions for screening for a candidate agent to inhibit pathologyassociated with MOG polypeptide-specific antibody binding to a MOGpolypeptide. These methods generally comprise the steps of:

incubating a mixture comprising: the antibody or a MOG-specific fragmentthereof, the MOG polypeptide, and a candidate agent,

under conditions whereby, but for the presence of said agent, theantibody or fragment thereof specifically binds the MOG polypeptide at areference affinity;

detecting the binding affinity of antibody or fragment thereof to theMOG polypeptide to determine an agent-biased affinity,

wherein a diminution of the agent-biased affinity with respect to thereference affinity indicates that said agent inhibits the binding of theantibody or fragment thereof to the MOG polypeptide and provides acandidate agent for inhibiting pathology associated with MOGpolypeptide-specific antibody binding to a MOG polypeptide.

In yet another embodiment, the invention provides polypeptidescomprising MOG-specific B- and T-cell epitopes, including polypeptidescomprising a fragment having N and C ends and consisting of residues28-36, 13-21, 67-73, 27-34 or 40-45 of human, rat or marmoset MOG (SEQID NO:1, SEQ ID NO:3 and SEQ ID NO:2, respectively), wherein thefragment is directly joined at at least one of the N and C-ends withother than natural human or marmoset MOG flanking residues. Suchpolypeptides are useful, for example in methods of inhibiting MOGpolypeptide-autoantibody binding, such as the general method comprisingthe step of contacting a mixture of a MOG and an antibody with apolypeptide, whereby the MOG-antibody binding is inhibited.

As used herein, the term “antibody” refers to recombined immune proteinssuch as T-cell antigen receptors and immunoglobulins, as well aschimeric, humanized or other recombinant antibodies. As used herein, theterm “antibody fragment” refers to fragments of antibodies such as Fab,Fab′, F(ab)₂, F(ab′)₂ and Fv or any combination thereof. Fv andfragments thereof may be monovalent or divalent. Fv is also known in theart as a minimal antibody fragment. Methods of making antibodyfragments, particularly F(ab′) are known in the art. (See for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1988), incorporated herein by reference). Forexample, F(ab), Fv, etc. can also be produced by recombinant technology.

As used herein, “other than natural human or marmoset MOG flankingresidues” refers to anything other than residues naturally flanking therecited peptides in the native proteins. For example, other than naturalflanking residues includes no flanking residues or flanking residuesdifferent from what naturally flanks the recited peptide.

MOG was originally identified by the mouse monoclonal antibody 8.18.C5,raised against rat cerebellar glycoproteins. It is a quantitativelyminor protein representing only 0.01 to 0.05% of the total myelinproteins and has no known function within the CNS. MOG is a member ofthe immunoglobulin (Ig) superfamily, with an immunoglobulin-like,extracellular domain comprised of 121 amino acids containing oneglycosylation site (Asn in position 31) and two highly hydrophobicregions that could represent transmembrane domains, for a total lengthof 224 amino acids. MOG is widely expressed on oligodendrocyte cellbodies and processes, especially on the outermost layers of the myelinsheaths, and may be more readily accessible to antibody attack thanintra-cytoplasmic MBP, or intra and inter-membranous proteolipidapoprotein (PLP). In all species studied including C. jacchus, thenon-glycosylated, recombinant extracellular domain of MOG (rMOG) whichis highly conserved, suffices for sensitizing animals for EAE. In oneaspect of the invention, we have identified minimal T-cell and B-cellepitopes, including residues 28-36, 13-21, 62-74, 27-34 or 40-45;natural human and rat MOG sequences (SEQ ID NO:3 and SEQ ID NO:1,respectively) are known in the art; natural marmoset MOG (SEQ ID NO:2)is identical to the human except for the following substitutions: 9S,13Q, 19A, 20A, 42S, 60E, 75D, 84K, 91P, 112Q, 137F, 148Y and 151H.

The immune response in autoimmune diseases may possess both cellular andhumoral components. Our data indicate that the following sequence ofevents leads to myelin destruction in CNS autoimmune demyelination:

1) Myelin vacuolation caused by soluble mediators (cytokines,antibodies, free radicals), and/or cellular cytotoxicity. A pattern ofintramyelinic edema similar to this has also been observed previously inthe CNS of rats intoxicated with tri-ethyl tin sulfate and,interestingly, these changes were reversible.

2) Transformation of vacuolated myelin into networks of small vesiclesseparated by 2-3 layers of altered myelin with a reduced periodicity(5-6 nm). This dramatic transformation appears to be associated with thedeposition of MOG-specific IgG and to reflect antibody-mediated damage,possibly due to complement activation, or antibody-dependentcytotoxicity mediated by macrophages that are invariably associated withvesicular myelin disruption. Conceivably, the initial vacuolar lesionrenders the myelin membranes accessible to an attack by autoantibodies.

3) Macrophage activation leading to receptor-mediated phagocytosis ofthe vesiculated myelin debris. This mechanism has been demonstratedpreviously in MS and in EAE with IgG serving as a ligand between themyelin debris and Fc receptors in clathrin-coated pits on the macrophagesurface. This stage of lesion pathogenesis, although antibody-mediated,may be independent of antibody specificity.

As just outlined above, for example, in MS the inflammatory component isT-cell mediated while the demyelinating component appears to be B-cellmediated. Thus, effective treatments should address both components.

The present invention provides compositions comprising theimmunodominant epitopes of MOG. The abolition of the peripheral T-cellresponse by a tolerization protocol to the extracellular portion ofrecombinant MOG (aa 1-125) (rMOG; rMOG is comprised of residues 1-125 ofthe extracellular amino terminus of MOG extended by MRGS at the NH₂ andASES(H)₆ at the COOH termini) provided the basis for the presentinventive epitope-derived peptide compositions. Mapping of the criticalMOG epitopes (including 26-38 and 64-72) was accomplished by cloningT-cells from rMOG-immunized animals and by analyzing T-cell and antibodyresponses to short peptides of MOG in rMOG immunized marmosets.

Mapping of the antibody response to MOG in C. jacchus indicates limitedheterogeneity of epitope recognition by autoantibodies. We haveidentified regions of MOG that are targeted by demyelinating antibodiesusing linear peptides. The native, serum polyclonal antibodies inrMOG-immunized marmosets are directed against 4 discrete epitopes alongthe amino acid sequence, aa 13-21, 28-34, 40-45, 65-74 or shortersequences, most of which are conserved sequences across species. Thesepeptides differ from those identified to date as antibody epitopes inrodents (aa 35-55), however they bind to antibodies present within thenetwork of vesiculated myelin in acute lesions of human MS as shown inthe Examples below. Because most antibodies generally recognizediscontinuous epitopes on proteins, our analysis methodology providesdetailed knowledge of the structure of MOG is needed to fully define theantigenic repertoire of demyelinating antibodies in C. jacchus andhumans. Combinatorial libraries were then made in order to generateF(ab′)₂ fragments with high affinity for MOG capable of competing withpathogenic IgG and of inhibiting complement-mediated and antibodydependent cellular cytotoxicity. These F(ab′)₂ fragments were testedalone and in combination with T-cell tolerogenic peptides for theirability to prevent and treat disease in C. jacchus.

A recently identified patient with a progressive spinal cord disorderassociated with an IgG monoclonal gammopathy reactive to MOG offered aunique example of the pathophysiologic consequences of an anti-MOGantibody response in a natural experiment. The human monoclonal antibodywas adoptively transferred into a C. jacchus with non-demyelinating EAE.Following adoptive transfer the marmoset developed demyelination.Transfer of human IgG in this species is well-tolerated and the blockingability of F(ab′)₂ fragments is demonstrated in the adoptive transfersystem. The antibody fragments retain their ability to recognizeantigenic epitopes yet lack the ability to activate complement or bindmacrophages, they coat the autoantigen such that the endogenousautoantibodies are unable to bind a pathological level.

In the preparation of the pharmaceutical compositions of this invention,a variety of vehicles and excipients and routes of administration may beused, as will be apparent to the skilled artisan. Representativeformulation technology is taught in, inter alia, Remington: The Scienceand Practice of Pharmacy, 19th ed., Mack Publishing Co., Easton, Pa.,1995; e.g. Goodman & Gilman's The Pharmacological Basis of Therapeutics,9^(th) Ed., 1996, McGraw-Hill.

EXAMPLES

The following examples are offered to illustrate this invention and arenot meant to be construed in any way as limiting in scope of thisinvention.

We show that antibodies against MOG that cause demyelination inmarmosets may be modified chemically and used as therapeutic tools tocompetitively block the binding of real-life, pathogenic antibody. Thisis achieved by enzymatic digestion, e.g., with pepsin, which cleaves theintact antibody into a large fragment (F[ab′]₂) that contains the sitesthat bind to the target antigen (MOG), and smaller fragments includingthe Fc portion, a portion of antibodies known to contain receptors forsystems such as complement and macrophages (also known to mediate manypathogenic or cytotoxic properties of antibodies). Thus, the F(ab′)₂retains the capacity to bind to MOG in the brain, but is devoid ofcapacity to Fc complement or activate macrophages and protects/masks MOGotherwise recognized by the pathogenic antibodies. In a particularexample, a pair of marmosets were first sensitized to EAE with MBP(non-demyelinating), then both given intravenous demyelinating antibody(mouse monoclonal 8.18.C5) against MOG. Simultaneously, one animal(control) received a placebo F(ab′)₂ injection, and the other receivedF(ab′)₂ fragments prepared from the same demyelinating antibody. Thecontrol animal showed aggravation of the clinical signs of EAE and theexperimental animal did not. The animals were sacrificed 3-5 days laterand histology of the brain and spinal cord obtained. The control animalhad evidence of demyelinating lesions, and the experimental animal hadlesions with inflammation (cellular infiltration) but no demyelination(no myelin destruction). This experiment shows that marmosets can beprotected from antibody mediated demyelination by MOG-specific F(ab′)₂fragments.

Complementary experiments of the above indicate that such therapeuticprinciple of F(ab′)₂ or F(ab′) fragments could be useful for human MS orrelated disorders: antibodies against MOG are intimately associated withactive lesions of MS where there is morphologic evidence for the ongoingdisintegration of myelin (see “Identification of autoantibodiesassociated with demyelination in multiple sclerosis”, below). In thiswork, the epitopes of MOG that are recognized by marmosets are alsodisclosed for the first time and this information is used to constructgold-conjugates as immunoprobes to identify the presence of MOG-specificautoantibodies in both primate and human tissues.

Induction of Marmoset EAE

EAE was induced in marmosets as described by Genain et al. (1999) NatureMedicine 5, 170-175. Six marmosets were actively sensitized with 50 to100 μg of recombinant rat MOG dissolved in 100 μl of phosphate-bufferedsaline and emulsified with an equal volume of complete Freund's adjuvant(CFA) containing 3 mg/ml killed Mycobacterium tuberculosis (h37Ra;DIFCO, Detroit, Mich.). The MOG/CFA emulsion was given intradermally atfour injection sites in the scapular and hip regions in a total volumeof 0.2 ml. On the day of immunization with MOG/CFA, 1×10¹⁰ inactivatedBordetella pertussis organisms in 2.5 ml of isotonic saline were givenintravenously and the dose repeated 2 days later. For comparison, 4marmosets sensitized with 200 mg of whole white matter (WM)/CFA and B.pertussis were examined between 18 and 39 days after immunization.MOG-sensitized marmosets were maintained for up to 93 days afterimmunization. Animals sensitized with either WM or MOG displayed signsof EAE within 21 days of immunization. The animals were sacrificed byintracardiac perfusion under deep anesthesia 18 to 93 days afterimmunization.

MS Tissues from Humans

Human CNS tissues were obtained from 3 subjects with MS by biopsy orautopsy (8 weeks, 11 years and 17 years after diagnosis). Patient 1 wasan 18-year old Caucasian woman with a 3-month history of acutelydeveloping right hemiplegia, sensory loss, and spasticity. Computedtomographic scanning revealed WM hypodensity in the leftparieto-occipital region. A brain biopsy was performed forneuropathological evaluation. The resultant diagnosis was activelydemyelinating, inflammatory, edematous lesions of recent origin, typicalof a fulminant inflammatory demyelinating condition, consistent withacute MS.

Patient 2 was a 31 year old Caucasian female with an 8-year history ofchronic progressive MS characterized by numbness and weakness of thelimbs, gait disturbance, urinary incontinence, tremor, nystagmus, andblurred vision. Terminally, the patient was wheel-chairbound, developedseizures and aspiration pneumonia, and died. An autopsy was performedwithin 1.5 hours of death. Neuropathological examination revealed apredominance of small (3-5 mm), disseminated, recent, intenselyinflamed, edematous, demyelinating lesions as well as larger, moreestablished plaques with fibrous astrogliosis and well-demarcated edges.

Patient 3 was a 34-year old Caucasian female with a history ofrelapsing-remitting MS for 10 years after initial diagnosis at age 20.The disease entered a chronic progressive course for the last 7 years ofher life. At the time of death, the patient presented with bilateraloptic atrophy, internuclear ophthalmoplegia, spastic paraparesis, andmoderate limb ataxia. The cause of death was respiratory failure. Anautopsy was performed 4 hours after death. Neuropathology of this caserevealed intensely inflammatory, edematous, actively demyelinatinglesions of recent origin, as well as active chronically demyelinatedlesions.

Tissue Preparation for Analysis

At the time of sampling, animals were sacrificed under pentobarbitalanesthesia by intracardiac perfusion with 200 ml of phosphate-bufferedsaline followed by 150 to 200 ml of cold PO₄-buffered 2.5%glutaraldehyde. Two MOG-sensitized marmosets were sampled during theacute phase of the disease (14-16 days after immunization), 3 were takenafter the acute phase, either during relapses or remission (23, 25 and27 days after immunization), and 1 was examined after two relapses at 93days after immunization. The 4 whole WM-sensitized animals were examinedat 18, 30, 30 and 39 days after immunization after acute onset butbefore relapse. From the glutaraldehyde-perfused animals, the CNS wasremoved and routine neuropathology performed on formalin postfixed,paraffin-embedded material stained with hematoxylin and eosin, LusolFast Blue (for myelin), and the Bodian silver technique (for axons).

For fine structural analysis of marmoset tissues, 1-mm slices were takenform optic nerve, cerebral hemispheres, cerebellum, brainstem, medulla,and spinal cord at C7, T3, L2, L5, L6, and L7. In addition, samples weretaken from spinal nerve roots and sciatic nerves. The slices ofglutaraldehyde-fixed brain tissue were trimmed as flat rectangles (˜4×6mm) and spinal cord was left as whole slices. From the 3 cases of MSdescribe in Example 2, small pieces of biopsy tissue or slices ofautopsied CNS material, 3 to 5 mm thick, were immersion-fixed for 4 to24 hours at 4° C., then cut into thin, 1-mm slices to 3 to 5 mm indiameter. Glutaraldehyde-fixed tissues were then postfixed inPO₄-buffered 1% OsO₄ for 1 hour on ice. Samples were dehydrated, clearedin propylene oxide, and embedded flat in epoxy resin. Thin (1 μm)sections of epoxy-embedded tissue were prepared for light microscopy(LM) and stained with toluidine blue or reacted for immunocytochemistry.For electron microscopy (EM), sections were placed on copper grids,contrasted with lead and uranium salts (lead citrate and uranylacetate), carboncoated, and scanned in a Siemens 101 or Hitachi H 600-S.

Ultrastructural Patterns of Demyelination are Identical in C. jacchusEAE and in Acute MS Plaques

CNS tissues from 6 C. jacchus marmosets with MOG-induced EAE and from 3human subjects with MS, all showing acute lesions, were examined byelectron microscopy (EM). In marmoset EAE, large demyelinated plaques upto several mm in diameter were disseminated throughout the CNS,invariably centered on venules and characterized by perivascularinflammation and a prominent margin along which many myelinated nervefibers displayed vacuolated myelin sheaths. This typical pattern ofmyelin vacuolation resulted from the enlargement of individual myelinsheaths due to interlamellar splitting and swelling, with the axondisplaced to one side surrounded by several layers of intact myelin.Micrographs showing the optic nerve from an animal with acute EAEinduced by immunization with 50 μg of recombinant rat MOG in adjuvant,sacrificed 3 days after onset of clinical signs demonstrated thepresence of large intramyelinic vacuoles at the perimeter of ademyelinated lesion, with axons surrounded by normal-appearing myelinsheaths elsewhere.

Between the lesion center and the margin was a broad zone ofdemyelination containing macrophages laden with myelin debris. The moststriking finding was the presence within the demyelinated zone of largenumbers of axons surrounded by aggregates of disrupted myelin rearrangedas an expanded network. These axons were displaced laterally as themembranous network gradually became dissociated from the axon and takenup by adjacent macrophages.

Demyelination of fibers in acute MS was structurally identical to thatseen in marmoset EAE, with the demyelinated axon lying within amembranous network of myelin. Elsewhere in the edematous parenchyma,free floating aggregates of myelin debris were common. Electronphotomicrographs of tissue taken from a subcortical white matter biopsyfrom an 18-year old female patient with an 8-week history of neurologicsigns, white matter hypodensity on MRI scan and a diagnosis of acute MSshowed myelin around axons transformed into a vesicular network similarto that described above. Fibrous astroglial processes, naked axons and areactive, ameboid microglial cell (below), were also identified. Highresolution analysis of the myelin networks in both marmoset EAE andhuman MS revealed vesicles surrounded by 2 to 3 layers of looselycompacted membranes with a reduced periodicity (5-6 nm) when compared tointact myelin in normal tissue.

MOG-Specific Autoantibodies are Associated with Myelin Vesiculation inthe C. jacchus EAE Lesion

MOG is a quantitatively minor myelin protein (less than 0.05% of totalmyelin proteins) with an immunoglobulin (Ig)-like extracellular domainthat is expressed in abundance on the outermost layer of myelin sheaths,which may render it accessible to antibody attack. Althoughautoantibodies against MOG have been shown to enhance demyelination inseveral EAE models, the detailed interactions between these antibodiesand myelin membranes has not been investigated. To identify the sites ofautoantibody binding within demyelinating lesions, we performedimmunocytochemistry on frozen and epoxy-embedded marmoset CNS tissuewith gold-labeled anti-human IgG antibody (cross-reactive with marmosetIgG) followed by silver enhancement.

For the demonstration of antigen-specific autoantibodies in marmoset andhuman MS tissue in situ, a selection of myelin-related and controlpeptides were directly coupled to immunogold and applied to tissuesections. Immunogold labeling was performed on ultra-thin sections offrozen or fixed tissues. Gold conjugates were prepared of (1) three MOGpeptides (amino acids [aa] 1-20, aa 21-40, and aa 41-60 of human MOG(SEQ ID NO:3)) with known encephalitogenic activity in marmosets; (2)one MOG peptide that has been shown not to be encephalitogenic inmarmosets (aa 101-120); (3) one human myelin basic protein (MBP) peptide(aa 82-101) that is encephalitogenic in marmosets and immunodominant inhumans with the DR2 haplotype; and, as a control, (4) a peptide of mouseserum albumin (MSA; aa 560-574). Peptides having human MOG subsequenceswere synthesized using standard Fmoc chemistry and purified (>95%) byHPLC (Research Genetics Inc., Huntsville, Ala.): MOG 1-20, MOG 21-40,MOG 61-80, MBP 82-101, and MSA 560-574. The gold conjugates weresynthesized by using monosulfo-N-hydroxy succinimide-Nanogold labelingreagent (particle diameter, 1.4 nm), according to the manufacturer'sinstructions (Nanoprobes, Stonybrook, N.Y.), followed by extensivedialysis to remove unreacted peptide. Immunoreactivity was detected on1-μm epoxy sections of marmoset spinal cord tissue and active MSlesions. For this, sections were etched with sodium ethoxide,equilibrated in PO₄-buffered saline containing 0.05% Triton X-100, andblocked with 10% normal rabbit serum. Sections were incubated withpeptide-immunogold conjugates (1:100 in buffer) for 2 hours at roomtemperature. After washing, detection was performed by using silverenhancement (Nanoprobes). Sections were counterstained with toluidineblue. For the detection of IgG, sections were reacted with gold-labeledanti-monkey or anti-human IgG (Nanoprobes) at 1:100.

As controls, sections were either pretreated with unlabeledencephalitogenic MOG peptides or MBP to block the reaction, reacted withunlabeled nonencephalitogenic MOG peptide (aa 101-120) beforeapplication of the gold conjugates, or treated with gold-labeledirrelevant antigens (histone or MSA) or irrelevant IgG (anti-goat). Thespecificity of the labeling with the gold conjugates of encephalitogenicMOG peptides was also assessed in western blots where the MOG proteinwas first reacted with immune marmoset serum. Full details of the testand control reagents used to determine the specificity of theimmunoreactivity can be found in Genain et al., Nature Med. 5:170-175(1999).

Sections from the lumbar region of the spinal cord from the animals wereobtained. The positive reactivity of vesiculated myelin around axons,indicated the presence of IgG. Non-demyelinating axons did not stain.Positive reactivity for IgG was specifically found over the vesiculatednetworks of disrupted myelin surrounding axons.

We next identified the target antigens bound by these immunoglobulins bythe application of immunogold-labeled conjugates of selected peptides ofMOG and myelin basic protein (MBP). These myelin antigens were directlylabeled with the gold particles on their primary NH₂ residues and wereused to detect antigen-specific autoantibody in situ. With thistechnique, three separate gold-conjugated peptides of MOG wereco-localized over the networks of disintegrating myelin sheaths in apattern similar to that observed for gold-conjugated anti-IgG.

These peptides contained the amino acid sequences of MOG recognized bydemyelinating antibodies that develop in serum of MOG-immunizedmarmosets (aa 1-20, aa 21-40 and aa 61-80). The gold-conjugated MOGpeptide (aa 21-40) used has a sequence conserved across species.MOG-reactive droplets were also seen in surrounding macrophages,indicating the presence of internalized myelin debris to which anti-MOGantibody was bound. Positive reactivity with the labeled antigenindicated the presence of MOG-specific antibody in situ on vesicularmyelin around axons and on myelin debris within the extracellular spaceand macrophages. Normal myelin (around the majority of the fibers) wasnot stained.

In contrast, gold-labeled conjugates of a peptide containing animmunodominant epitope of human MBP (aa 82-101, conserved across primatespecies) and of a control peptide of mouse serum albumin (MSA, aa560-574), failed to label myelin membranes or macrophages. Thus, thevesiculated myelin networks are unstained by either the gold-conjugatedpeptide of MBP or the gold-conjugated peptide of MSA.

These observations demonstrate in this non-human primate model of EAEthat antibodies specific to MOG are in direct contact with thedisintegrating myelin membranes and indicate that formation of thevesiculated membranous networks resulted from lytic attack by theseautoantibodies.

MOG-Specific Autoantibodies are Associated with Myelin Vesiculation inLesions of Acute Human MS

We next investigated with similar immunogold labeling the presence ofMOG- and MBP-specific autoantibodies in CNS tissue obtained at biopsy orautopsy from patients with MS. As in marmoset EAE, gold-conjugatedanti-IgG labeled the membranous myelin networks around singledemyelinating axons, along with droplets of myelin debris scatteredthroughout the parenchyma. IgG is localized along the disintegratedmyelin sheath of an axon cut in longitudinal section; cytoplasm of anhypertrophied astrocyte; tangential section of an oligodendrocyte.Densely stained IgG-coated myelin debris are visible in the parenchymaand in 3 macrophages (probably ameboid microglia). In addition,occasional plasma cells showed positive staining by anti-IgG. With theimmunogold-labeled myelin antigen conjugates, vesiculated myelinnetworks were intensely stained by gold-conjugated-MOG peptides, and toa lesser extent by gold-conjugated MBP but not by MSA.

IgG-myelin complexes labeled with gold-conjugates of MOG and MBP werealso present in macrophages but not in astrocytes or oligodendrocytes.No MOG- or MBP-labeled plasma cells were encountered. Reactivity withgold-conjugates was not observed in normal appearing MS white matter oraround perivascular inflammatory cuffs. In marmosets immunized againstwhole myelin, a similar pattern of both anti-MOG and anti-MBP Igdeposition was observed. CNS tissue from amyotrophic lateral sclerosis,another neurologic disorder associated with white matter damage andmacrophage activity, failed to show myelin antigen-specific immunogoldreactivity. These findings directly identify MOG-specific antibodies inactively demyelinating lesions of human MS, indicating that, as inMOG-induced EAE, these autoantibodies play a causal role in theformation of small vesicles in the disrupted myelin sheaths. Soluble andB-cell surface Ig with anti-MBP specificity have been described in MSbrain tissue, and in the current study, MBP-specific Ig was localizedwithin the vesiculated myelin networks in MS lesions. Although anti-MBPantibodies have not been shown experimentally to initiate demyelinatingpathology, these autoantibodies can mediate separate pathogenicmechanisms such as receptor-mediated phagocytosis by macrophages and/orpresentation of myelin autoantigens to specific T-cells.

It is noteworthy that autoantibodies appear to be bound exclusively tothe small vesicles that characterize the stage of completedisintegration of the myelin membranes, and to the myelin debris presenteither in the extracellular space or in phagocytic cells. Interestingly,similar but less extensive vesiculation of myelin was reported inearlier studies of rodent EAE where it was perceived as a transientearly phenomenon. However, in the marmoset where lesion formation isprotracted and ever expanding, the disrupted myelin was foundconsistently. The large scale vacuolation of myelin at the lesion marginamong normally myelinated fibers occurred in the absence of significantlocal cellular infiltration or IgG deposition, and has also beenreported at the edge of active MS lesions. This change in the myelinstructure could be mediated by soluble factors diffusing from the centerof the demyelinating plaque or from activated glial cells at the edge ofthe lesion. Morphologic changes similar to these large vacuoles havebeen reported in myelinated CNS cultures exposed to TNF-alpha and to alesser degree, in cultures exposed to serum from animals with EAE andfrom subjects with MS.

Many of the therapeutic approaches targeting pathogenic T-cell responsesin EAE models have not yet translated into successful treatment forhuman MS, perhaps suggesting that other components of the immune systemneed to be taken into account. B-cell responses appear to be a keyfactor for severity of clinical disease and pathology in C. jacchus EAE.The current results underscores the role of autoantibodies in thewidespread destruction of myelin in MS, and emphasizes that in diseasesthat are initiated by T-cell responses, antibodies against criticalantigens of the target organ are essential for development ofirreversible tissue damage.

In vivo Administration of MOG-Specific F(ab′)₂ Fragments

Marmosets were administered 1 mg MBP in CFA containing B. Pertussis atDay 0 inducing non-demyelinating EAE. On Day 21 the animals wereadministered intravenously 0.17 mmol/kg of the F(ab′)₂ from either8.18.C5, a murine monoclonal anti-MOG antibody, or an anti-Influenza-A(control) antibody, then administered 0.17 mmol/kg 8.18.C5 antibodyfollowed by a second intravenous administration of the appropriateF(ab′)₂ for two hours. The animals were euthanized on day 35. Tissuesamples were prepared as described in Example 3. Using high resolutionmicroscopy and immunogold-labeled peptides of myelin antigens capable ofdetecting antigen-specific antibodies in situ, we have identifiedautoantibodies specific for myelin/oligodendrocyte glycoprotein (MOG)around individual demyelinating axons in acute lesions of both human MSand marmoset EAE, where they appear directly responsible for thedisintegration of myelin sheaths. Animals treated with control F(ab′)₂fragments, i.e., directed against influenza antigen, revealed largedemyelinating plaques in the cervical spinal cord, and animals treatedwith MOG-specific F(ab′)2 fragments showed that the demyelinatingactivity of anti-MOG antibody in marmosets is dependent on intact Fcfragment function, as it can be competitively blocked in vivo byadministration of MOG-specific F(ab′)₂ fragments. These findingsunderscore the role of myelin-specific autoantibodies in the widespreaddestruction of myelin in MS and provide a basis for protective therapyin CNS demyelinating disorders.

Encephalitogenic Epitope Determination (MOG) in MS-Like Marmoset EAE

In C. jacchus marmosets with demyelinating EAE induced with recombinantrat MOG (rMOG: extracellular domain aa 1-125), the fine specificities ofT-cell reactivity (proliferative responses in PBMC) and B-cellreactivity (serum antibody) to MOG were serially studied usingoverlapping 15-mer PIN-peptides (offsets of 1 and 3), corresponding toamino-acid sequences of both rat and human MOG (SEQ ID NO:1 and SEQ IDNO:3, respectively)(Chiron Mimotopes, San Diego, Calif.). Results: Allanimals studied (n=6) had a prominent and sustained T-cell responserestricted to aa 27-36, a sequence totally conserved across species. Asingle marmoset responded to a second T-cell epitope located within aa62-72. Serum antibody responses (n=10) mapped to 4 different regions ofMOG including 2 major epitopes, aa 13-21 and aa 62-74 (100% and 60% ofanimals, respectively) and additional epitopes were identified in someanimals (aa 28-36 and 40-45). No epitope spreading was observed eitherfor T-cells or antibodies in animals with relapsing EAE that weremonitored for up to 93 days. Conclusions: Encephalitogenic responses toMOG in MS-like, marmoset EAE appear restricted to a limited number ofB-cell and T-cell epitopes. These findings demonstrate feasibility ofspecific immunotherapy in human MS.

Detecting B-cells with Surface Bound Antibodies

Early in the immune response B-cells have on their surfaceimmunoglobulins that may specifically react with antigens. The B-cellimmunoglobulin reacting with a self-antigen may be the first step in anautoimmune disease. Thus, early detection of autoantibodies on thesurface of B-lymphocytes may provide the means to design a method oftreatment before the onset of symptoms.

B-cells constitute about 3-5% of lymphocytes and were positivelyselected from freshly isolated peripheral blood mononuclear cells (PBMC)obtained from C. jacchus marmosets with MOG-induced EAE, from humanswith MS and from healthy controls using anti-CD 19 coated beads.Antibodies to any other suitable B-cell marker may be used. Slidescontaining 2×10⁵ B-cells (>98% purity) were fixed with 1% glutaraldehydeand washed. Alternatively, unfixed cells may be used. The isolatedB-cells were then incubated with labeled immunogold conjugates of amixture of eleven 20-mer overlapping peptides corresponding to thesequence of the NH₂ terminus of human MOG (1-120 (of SEQ ID NO:3));identical B-cell preparations were labeled with control polypeptidescorresponding to the sequence of histone or MBP peptides. Slides wereenhanced with silver and labeled B-cells were counted by two differentblind observers.

B-cells expressing MOG-specific surface immunoglobulins were easilydetected with the gold-conjugated MOG peptides in PBMC fromMOG-immunized marmosets (n=8). In these animals which are known todevelop serum anti-MOG antibodies, circulating MOG-specific B-cellsoccurred at a frequency of about 1:500 to about 1:2,000, increased from0-1:10,000 in healthy, unimmunized marmosets (n=8). Unlabeled MOGpeptides added in excess completely inhibited labeling andgold-conjugated control protein failed to label any B-cell. In humans,circulating MOG-specific B-cells could be detected in 8 of 17 MSpatients (47%) and 9 of 18 healthy controls (50%). The frequency ofthese autoreactive B-cells ranged from about 1:11,000 to about 1:200,000B-cells, with the highest frequencies observed in two patients withrelapsing-remitting MS (1:16,000 and 1:11,000, respectively).

This immunogold assay sensitively detects MOG-specific B-cells inperipheral blood. This assay has a sensitivity of about 1:500,preferably of about 1:2,000, more preferably about 1:10,000, even morepreferably about 1:15,000, and most preferably about 1:200,000. Inhumans, autoreactive B-cells can be detected in approximately 50% ofindividuals, and are equally present in MS patients and controls. Thissuggests that the presence of anti-MOG antibodies in the nervous systemof individuals with MS is not associated with a major expansion ofMOG-reactive B-cells in the peripheral blood. The high frequency ofMOG-reactive B-cells observed in PBMC provides new support for thehypothesis that MOG is an important autoantigen in humans.

In the Examples above, MOG-specific antibodies were exclusivelylocalized to areas where the transformation of compact myelin into smallvesicles around single demyelinating axons occurred, and to myelindebris either floating in the CNS parenchyma or internalized byphagocytic cells (macrophages and microglia). Thus, in addition to adirect lytic attack on myelin and oligodendrocytes, these antibodies canalso be responsible for receptor-mediated phagocytosis by macrophages,or antibody-dependent cellular cytotoxicity, which have long beenrecognized as possible effector mechanisms of myelin damage. Ourexperiments using passive transfer of antibody in the C. jacchus systemhave shown that it is possible to competitively block pathogenic effectsof the monoclonal anti-MOG antibody 8.18.C5 by in vivo administration ofpurified 8.18.C5-F(ab′)₂ fragments, indicating that intact Fc fragmentsand not the MOG-specific CDR3 sequence themselves mediate the damage tomyelin. Based on these findings, analogs or competitive inhibitors ofantibody binding that are devoid of toxic effects on myelin provide arational approach for therapy in EAE and related demyelinatingdisorders. Thus, the present invention utilizes compositions ofautoantigen epitopes, anti-autoantigen antibody fragments orcombinations thereof to effectuate treatment of demyelinating autoimmunediseases.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims. Accordingly,it is intended that the scope of the present invention be limited solelyby the scope of the following claims, including equivalents thereof.

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What is claimed is:
 1. A method of inhibiting myelin oligodendrocyteglycoprotien (MOG)-antibody binding comprising the step of contacting amixture of a MOG and an antibody with a fragment having N and C ends andconsisting of residues 28-36, 13-21, 67-73, 27-34 or 40-45 of human MOG(SEQ ID NO:3), rat MOG (SEQ ID NO:1) or marmoset MOG (SEQ ID NO:2),whereby the MOG-antibody binding is inhibited.
 2. A method according toclaim 1, wherein the fragment consists of residues 28-36 of human MOG(SEQ ID NO:3).
 3. A method according to claim 1, wherein the fragmentconsists of residues 13-21 of human MOG (SEQ ID NO:3).
 4. A methodaccording to claim 1, wherein the fragment consists of residues 67-73 ofhuman MOG (SEQ ID NO:3).
 5. A method according to claim 1, wherein thefragment consists of residues 27-34 of human MOG (SEQ ID NO:3).
 6. Amethod according to claim 1, wherein the fragment consists of residues40-45 of human MOG (SEQ ID NO:3).
 7. A method according to claim 1,wherein the fragment consists of residues 28-36 of human MOG (SEQ IDNO:2).
 8. A method according to claim 1, wherein the fragment consistsof residues 13-21 of human MOG (SEQ ID NO:2).
 9. A method according toclaim 1, wherein the fragment consists of residues 67-73 of human MOG(SEQ ID NO:2).
 10. A method according to claim 1, wherein the fragmentconsists of residues 27-34 of human MOG (SEQ ID NO:2).
 11. A methodaccording to claim 1, wherein the fragment consists of residues 40-45 ofhuman MOG (SEQ ID NO:2).